J. Phy.iol. (1975), 253, pp. 411-428 With 4 text-ftgurem Printed in Great Britain
411
EFFECTS OF ANGIOTENSIN II ON FLUID TRANSPORT, TRANSMURAL POTENTIAL DIFFERENCE AND BLOOD FLOW BY RAT JEJUNUM IN VIVO
BY JENNIFER E. BOLTON, K. A. MUNDAY, B. J. PARSONS AND BARBARA G. YORK From the Department of Physiology and Biochemistry, Medical and Biological Sciences Building, University of Southampton, Southampton S09 3TU
(Received 3 March 1975) SUMMARY
1. A method has been described for the measurement of fluid transport by rat jejunum in vivo over two consecutive 30 min periods. 2. Subpressor infusion rates of angiotensin (0.59 ng/kg per minute) stimulate fluid transport, while high (pressor) infusion rates (590 ng/kg per minute) inhibit fluid absorption. 3. Both the inhibitory and stimulatory effects of angiotensin on fluid transport are not accompanied by any change in the transmural p.d., total blood flow to the jejunum or distribution of blood flow within the wall of the jejunum. 4. These results are discussed in relation to the mechanism of action of angiotensin on fluid transport and its role in sodium and water homoeostasis. INTRODUCTION
It is generally assumed that aldosterone is the major hormone involved in the control of sodium homoeostasis, although it is becoming increasingly evident that angiotensin must also be considered as a physiologically important sodium retaining hormone apart from its proposed role (Kaplan & Bartter, 1962) in the control of aldosterone secretion. The rate of renin secretion, and consequently the circulating angiotensin concentration, is dependent on the sodium status of the animal; sodium depletion consistently elevates plasma renin levels whereas sodium loading is usually associated with a fall in the plasma renin concentration (Vander, 1967). Furthermore, physiological concentrations of angiotensin have been shown to stimulate sodium transport by a range of mammalian transporting epithelia. For example angiotensin, at low concentrations,
JENNIFER E. BOLTON AND OTHERS enhances sodium transport by in vitro preparations of rat jejunum (Crocker & Munday, 1970), rat colon (Davies, Munday & Parsons, 1970) and rat kidney (Munday, Parsons & Poat, 1971). Similarly, the kidney in vivo responds to low infusion rates of the hormone with an antinatriuresis, but it is controversial whether this is due to direct stimulation of tubular sodium re-absorption mechanisms (Barraclough, Jones & Marsden, 1967) or is secondary to changes in intrarenal blood flow (Bonjour & Malvin, 1969). Much of the evidence implicating angiotensin as a salt retaining hormone has been obtained from studies carried out on in vitro intestinal preparations and, as such, may be subject to criticism. Consequently, the following experiments were undertaken to investigate the effects of intravenous infusions of angiotensin on fluid transport, and thus indirectly sodium transport, by rat jejunum in vivo. 412
METHODS
Animal8 Male albino Wistar rats, weighing approx. 300 g, were starved overnight before use.
Experimental procedure The rats were anaesthetized with pentobarbitone sodium (100 mg/100 g body wt.) i.P. after which the trachea, left common carotid artery and both femoral veins were cannulated. A pressure transducer was connected to the carotid cannula in order to continuously monitor arterial blood pressure. The femoral vein cannulae were then independently attached to separate syringes in a Palmer constant-rate infusion pump so that either 09 % NaCl (containing 50 u. heparin/ml.) could be infused into one femoral vein or, alternatively, angiotensin in isotonic heparinized saline could be infused into the second femoral vein. The rate of infusion was always 1*0 ml./hr apart from the first min of any infusion when it was increased to 01 ml./min in order to establish the potency of the cannula. A mid line abdominal incision was made through the skin and muscle layers in order to expose the viscera. The proximal end of the jejunum was found at the ligament of Treitz and ligatured, after which a second ligature was tied approx. 20 cm distal to the first. The resulting isolated segment of proximal jejunum was then thoroughly washed with isotonic saline and gently emptied. Finally, two new ligatures were tied around the central region of the isolated segment of proximal jejunum to give a closed sac approx. 15 cm in length. Great care was taken with the placing of all ligatures so that the blood supply to the sac of jejunum was not
impaired. Measurement of fluid transport Approximately 5 ml. Krebs bicarbonate buffer (Krebs & Henseleit, 1932) containing [3H]inulin (200,000 d.p.m./ml.), as a non-absorbable marker, was injected into the sac through a small diameter (26 s.w.G.) hypodermic needle. The jejunum was gently massaged to mix the sac contents, and at 0 min a 0-1 ml. sample was removed through a hypodermic needle and assayed for tritium. Immediately after
ANGIOTENSIN ON INTESTINAL TRANSPORT
413
this, the sac of jejunum was returned to the abdominal cavity and saline infused into one of the femoral veins at a rate of 1 ml./hr. After 30 min, the jejunum sac was again exposed and massaged to mix the contents. A second 0-1 ml. sample of the sac contents was removed and assayed for tritium. The sac was returned to the abdomen, and at the same time the saline infusion was stopped and replaced by an infusion of angiotensin in saline, at the same rate, through the other femoral vein for a second 30 min period. Finally, at the end of 60 min the contents of the sac were mixed and a third sample collected and assayed for radioactivity. The sac of jejunum was removed from the animal by cutting the intestine immediately beyond each ligature and weighed. It was then emptied, blotted by a standardized procedure on filter paper (Whatman No. 50) and re-weighed to obtain the volume of the contents and the weight of the sac. The precise volume of buffer injected into the sac at the beginning of the experiment and the volume of subsequent samples were obtained by weighing. Inulin is a non-absorbable marker so that, following the absorption of fluid from the jejunum sac, there is an increase in the inulin concentration. From the volume of fluid in the sac at 0 min and the concentrations of inulin in the sac contents at 0, 30 and 60 min and the volume of all samples removed, fluid absorption from the sac during two consecutive 30 min periods was calculated. A second procedure for assessing fluid transport was routinely carried out as a check on the measurement of fluid transport by the inulin method. In this, fluid transport over the whole 60 min period was calculated from the volume of the contents of the jejunum sac at 0 and 60 min and the total volume of all samples removed from the sac during the experiment.
Measurement of transmural potential difference In some experiments the transmural potential difference, p.d., of the jejunum sac was measured. A length of 30 pp Portex tubing containing 3 M-KC1 in 4 % agar gel was used as the mucosal electrode and ligatured into the distal end of the jejunum sac. An i.P. agar-saline bridge was used as the serosal electrode. Both electrodes were connected, via calomel half-cell electrodes, to a Vibron electrometer which was used to measure the p.d. across the mucosal and serosal electrodes. The transmural p.d. was continuously monitored on a recorder. The asymmetry between the intraluminal and serosal electrodes was always checked before experiments by placing their tips together in isotonic saline and did not exceed 0-3 mV. The mean transmural p.d. was obtained from the mid-point of the best straight line through the p.d. recording. Measurement of blood flow to the jejunum Blood flow to the jejunum was obtained from the product of cardiac output and the percentage of cardiac output passing to the jejunum. Animals were prepared as described previously for measurements of fluid absorption and jejunum blood flow determined during the infusion of saline or angiotensin in saline. (a) Cardiac output. The thermal dilution method of Fegler (1954) was used. The left common carotid artery was cannulated and a thermistor inserted so that the tip lay just inside the aortic arch. Precisely 0- ml. of 0-85 g/100 ml. saline, at room temperature, was injected every 5 min through a cannula placed in the left jugular vein. Cardiac output was calculated from the arterial temperature-dilution curves obtained at 5 min intervals over a 30 min period during the infusion of saline and during a second 30 min period whilst saline or angiotensin in saline was infused through the femoral veins at a rate of 1 ml./hr.
414
JENNIFER E. BOLTON AND OTHERS
(b) Percentage of cardiac output distributed to the jejunum. Two independent methods were used to measure this parameter. The first method was essentially that described by Sasaki & Wagner (1971) with the exception that macro-aggregated iodinated (1311) human serum albumin was used as an alternative to microspheres. The majority of these particles had a diameter between 20 and 50 ,tm and consequently were trapped by intestinal capillaries so that the percentage of intracardiac administered macro-aggregated albumin collected in the jejunum could be taken as a measure of the percentage of cardiac output delivered to the jejunum. Nylon 00 tubing, filled with heparanized saline and attached to a blood pressure transducer, was used to cannulate the right carotid artery. The cannula was inserted into the left ventricle and its position verified by the large increase in the pulse pressure as the cannula passed the aortic valves. Saline was infused i.v. for 15 or 45 min after which 0-2 ml. (1 /,cc) macroaggregated albumin was injected into the left ventricle. When the effects of angiotensin were studied, saline was infused for 30 min followed by angiotensin for 15 min after which the macro-aggregated albumin was injected. The sac of jejunum was removed from the animal 10 min after the injection of macro-aggregated albumin, opened, damp blotted, weighed and assayed for 131I in a y-well scintillation counter. The second method was that of Sapirstein (1958) which depends on the observation that 86Rb is almost completely cleared from the plasma during its passage through an organ. In these experiments, 0-2 ml. (1 #uc) 86Rb Cl was injected into a femoral vein and 45 see later the intestine removed, blotted, weighed and assayed for 86Rb in a y-well scintillation counter.
Radioactivity assay Each 0-1 ml. sample of intestinal contents, containing [H3]inulin, was added to a counting vial containing 10 ml. scintillation fluid (0-8 % butyl PBD in 1: 1, v /v methanol :toluene) and counted in a Phillips automatic scintillation counter. Intestines, containing 86Rb or l3lI, were dissolved in conc. HNO4, made up to 5 ml. with distilled water and counted in a y-well scintillation counter. All counts were corrected for background and quenching to give d.p.m. Expression of results Fluid absorption is defined as the loss of fluid from the jejunum sac during the experiment and is recorded as ml./30 min per gram wet weight of sac. There is no significant change in the water content of the jejunum following the infusion of angiotensin, the wet/dry wt. ratio being 5-9 ± 0-4 (5) following the infusion of saline and 6-1 ± 0-3 (4) and 6-2 ± 0-7 after the infusion of 0-59 and 590 ng angiotensin/ kg per minute respectively. Transmural p.d. is taken as the mean p.d. recorded over a 30 min period. Student's t test was used to determine the significance of differences between means.
Materials Angiotensin (Hypertensin, Ciba) was diluted with sterile saline to a concentration of 10 jug/ml. and stored in sealed siliconized ampoules. Immediately before use, the angiotensin was diluted to a suitable concentration in isotonic saline. [3H]inulin 86RbCl and 13II-labelled macro-aggregated albumin were obtained from the Radiochemical Centre, Amersham.
ANGIOTENSIN ON INTESTINAL TRANSPORT
415
RESULTS
The use of non-absorbable markers to measure fluid transport has been well documented. In a comprehensive study, Miller & Schedl (1970) showed that less than 2 % of inulin was lost from an intestinal perfusate during a 3 hr test and concluded that this material is suitable for measuring fluid transport in preparations of small intestine. The effectiveness of inulin as a non-absorbable marker was confirmed in the modified preparation of jejunum used in these experiments. Fluid transport was
30O 4.1 41
4.1
0
0. co
CL C
-
L
4J
1-0io
120 60 90 Time (min) Fig. 1. The rate of fluid absorption, as a function of time, from sacs of rat jejunum in vivo. Each point represents the mean of two observations.
30
determined in a series of animals by both the inulin method and by the direct technique of measuring the loss of weight of the sacs. Over a period of 60 min, in eight preparations, fluid transport measured by the inulin method (1.48 + 0-10 ml./g wet wt. jejunum per hour) was not significantly different from that obtained by direct weighing (1.59 + 0'13 ml./g wet wt. jejunum per hour). Furthermore, in the presence of 30 mg/tOO ml. carrier inulin, 93-6 + 4-1 0/ of the inulin injected into the sac was recovered at the end of the experiment. The suitability of the preparation was further tested by measuring T.1
PHY 253
416
JENNIFER E. BOLTON AND OTHERS
V-
0 0
0 0
V
V
v
C)
C0
o0 CO
0
0-
.5
.--
0
0
COm
10
IV 0
0 C) 0
0l
10
10-
I-
.0 -Q
0
00 0
+1
+1
+1 0
0
W-
CO
0
pI fQ
+1 0 0
o
.--
444
.-10
t-
0 0
co 10
+l
+1
C6
C>
(M
'.
0
a44 0
CO
0 O
.;Z
W4 4
t-
~00
to
w
0
ODw
0 m
> C)
_
I
EM¢
m
o
0;0
3
+I
.--
~~~~~0 0
I-
02
w
-44
._
o -
Q
e
(D
PIZ
P.-
o MD
OD
(ID
GQ°
2
ANGIOTENSIN ON INTESTINAL TRANSPORT 417 the rate of fluid transport for varying periods to a maximum of 2 hr. The results of these experiments are given in Fig. 1 and show that the rate of fluid transport is constant for the first 60 min. After 60 min the rate of fluid absorption is reduced, presumably as a consequence of the small volume of fluid remaining in the sac and available for transport from this time. Having established the validity of using inulin as a non-absorbable marker to measure fluid transport from isolated sacs of rat jejunum and having shown that absorption is constant over a 60 min period, the following experiments were carried out to study the effects of angiotensin 10 4._
~.200
0
:g 150 0
L
_
Fig. 2. Dose dependent actions of angiotensin on fluid transport by rat jejunum in vivo. Angiotensin was infused during the second period and fluid transport is expressed as rate of transport in the second period as a percentage of that in the first (control) period. Bars represent s.E. of mean.
On fluid transport by this preparation. Fluid transport was measured over two consecutive 30 min periods. Isotonic saline was infused ix.V during the first 30 min period while saline or angiotensin in saline was
infused during the second period. In this way the first 30 min absorption period was used as a control for the second period. The results of these experiments are presented in Table 1. There was no significant difference between fluid absorption in the first and second 30 min periods in those experiments where saline was infused continuously, confirming the results presented in Fig. I that
411
EFFECTS OF ANGIOTENSIN II ON FLUID TRANSPORT, TRANSMURAL POTENTIAL DIFFERENCE AND BLOOD FLOW BY RAT JEJUNUM IN VIVO
BY JENNIFER E. BOLTON, K. A. MUNDAY, B. J. PARSONS AND BARBARA G. YORK From the Department of Physiology and Biochemistry, Medical and Biological Sciences Building, University of Southampton, Southampton S09 3TU
(Received 3 March 1975) SUMMARY
1. A method has been described for the measurement of fluid transport by rat jejunum in vivo over two consecutive 30 min periods. 2. Subpressor infusion rates of angiotensin (0.59 ng/kg per minute) stimulate fluid transport, while high (pressor) infusion rates (590 ng/kg per minute) inhibit fluid absorption. 3. Both the inhibitory and stimulatory effects of angiotensin on fluid transport are not accompanied by any change in the transmural p.d., total blood flow to the jejunum or distribution of blood flow within the wall of the jejunum. 4. These results are discussed in relation to the mechanism of action of angiotensin on fluid transport and its role in sodium and water homoeostasis. INTRODUCTION
It is generally assumed that aldosterone is the major hormone involved in the control of sodium homoeostasis, although it is becoming increasingly evident that angiotensin must also be considered as a physiologically important sodium retaining hormone apart from its proposed role (Kaplan & Bartter, 1962) in the control of aldosterone secretion. The rate of renin secretion, and consequently the circulating angiotensin concentration, is dependent on the sodium status of the animal; sodium depletion consistently elevates plasma renin levels whereas sodium loading is usually associated with a fall in the plasma renin concentration (Vander, 1967). Furthermore, physiological concentrations of angiotensin have been shown to stimulate sodium transport by a range of mammalian transporting epithelia. For example angiotensin, at low concentrations,
JENNIFER E. BOLTON AND OTHERS enhances sodium transport by in vitro preparations of rat jejunum (Crocker & Munday, 1970), rat colon (Davies, Munday & Parsons, 1970) and rat kidney (Munday, Parsons & Poat, 1971). Similarly, the kidney in vivo responds to low infusion rates of the hormone with an antinatriuresis, but it is controversial whether this is due to direct stimulation of tubular sodium re-absorption mechanisms (Barraclough, Jones & Marsden, 1967) or is secondary to changes in intrarenal blood flow (Bonjour & Malvin, 1969). Much of the evidence implicating angiotensin as a salt retaining hormone has been obtained from studies carried out on in vitro intestinal preparations and, as such, may be subject to criticism. Consequently, the following experiments were undertaken to investigate the effects of intravenous infusions of angiotensin on fluid transport, and thus indirectly sodium transport, by rat jejunum in vivo. 412
METHODS
Animal8 Male albino Wistar rats, weighing approx. 300 g, were starved overnight before use.
Experimental procedure The rats were anaesthetized with pentobarbitone sodium (100 mg/100 g body wt.) i.P. after which the trachea, left common carotid artery and both femoral veins were cannulated. A pressure transducer was connected to the carotid cannula in order to continuously monitor arterial blood pressure. The femoral vein cannulae were then independently attached to separate syringes in a Palmer constant-rate infusion pump so that either 09 % NaCl (containing 50 u. heparin/ml.) could be infused into one femoral vein or, alternatively, angiotensin in isotonic heparinized saline could be infused into the second femoral vein. The rate of infusion was always 1*0 ml./hr apart from the first min of any infusion when it was increased to 01 ml./min in order to establish the potency of the cannula. A mid line abdominal incision was made through the skin and muscle layers in order to expose the viscera. The proximal end of the jejunum was found at the ligament of Treitz and ligatured, after which a second ligature was tied approx. 20 cm distal to the first. The resulting isolated segment of proximal jejunum was then thoroughly washed with isotonic saline and gently emptied. Finally, two new ligatures were tied around the central region of the isolated segment of proximal jejunum to give a closed sac approx. 15 cm in length. Great care was taken with the placing of all ligatures so that the blood supply to the sac of jejunum was not
impaired. Measurement of fluid transport Approximately 5 ml. Krebs bicarbonate buffer (Krebs & Henseleit, 1932) containing [3H]inulin (200,000 d.p.m./ml.), as a non-absorbable marker, was injected into the sac through a small diameter (26 s.w.G.) hypodermic needle. The jejunum was gently massaged to mix the sac contents, and at 0 min a 0-1 ml. sample was removed through a hypodermic needle and assayed for tritium. Immediately after
ANGIOTENSIN ON INTESTINAL TRANSPORT
413
this, the sac of jejunum was returned to the abdominal cavity and saline infused into one of the femoral veins at a rate of 1 ml./hr. After 30 min, the jejunum sac was again exposed and massaged to mix the contents. A second 0-1 ml. sample of the sac contents was removed and assayed for tritium. The sac was returned to the abdomen, and at the same time the saline infusion was stopped and replaced by an infusion of angiotensin in saline, at the same rate, through the other femoral vein for a second 30 min period. Finally, at the end of 60 min the contents of the sac were mixed and a third sample collected and assayed for radioactivity. The sac of jejunum was removed from the animal by cutting the intestine immediately beyond each ligature and weighed. It was then emptied, blotted by a standardized procedure on filter paper (Whatman No. 50) and re-weighed to obtain the volume of the contents and the weight of the sac. The precise volume of buffer injected into the sac at the beginning of the experiment and the volume of subsequent samples were obtained by weighing. Inulin is a non-absorbable marker so that, following the absorption of fluid from the jejunum sac, there is an increase in the inulin concentration. From the volume of fluid in the sac at 0 min and the concentrations of inulin in the sac contents at 0, 30 and 60 min and the volume of all samples removed, fluid absorption from the sac during two consecutive 30 min periods was calculated. A second procedure for assessing fluid transport was routinely carried out as a check on the measurement of fluid transport by the inulin method. In this, fluid transport over the whole 60 min period was calculated from the volume of the contents of the jejunum sac at 0 and 60 min and the total volume of all samples removed from the sac during the experiment.
Measurement of transmural potential difference In some experiments the transmural potential difference, p.d., of the jejunum sac was measured. A length of 30 pp Portex tubing containing 3 M-KC1 in 4 % agar gel was used as the mucosal electrode and ligatured into the distal end of the jejunum sac. An i.P. agar-saline bridge was used as the serosal electrode. Both electrodes were connected, via calomel half-cell electrodes, to a Vibron electrometer which was used to measure the p.d. across the mucosal and serosal electrodes. The transmural p.d. was continuously monitored on a recorder. The asymmetry between the intraluminal and serosal electrodes was always checked before experiments by placing their tips together in isotonic saline and did not exceed 0-3 mV. The mean transmural p.d. was obtained from the mid-point of the best straight line through the p.d. recording. Measurement of blood flow to the jejunum Blood flow to the jejunum was obtained from the product of cardiac output and the percentage of cardiac output passing to the jejunum. Animals were prepared as described previously for measurements of fluid absorption and jejunum blood flow determined during the infusion of saline or angiotensin in saline. (a) Cardiac output. The thermal dilution method of Fegler (1954) was used. The left common carotid artery was cannulated and a thermistor inserted so that the tip lay just inside the aortic arch. Precisely 0- ml. of 0-85 g/100 ml. saline, at room temperature, was injected every 5 min through a cannula placed in the left jugular vein. Cardiac output was calculated from the arterial temperature-dilution curves obtained at 5 min intervals over a 30 min period during the infusion of saline and during a second 30 min period whilst saline or angiotensin in saline was infused through the femoral veins at a rate of 1 ml./hr.
414
JENNIFER E. BOLTON AND OTHERS
(b) Percentage of cardiac output distributed to the jejunum. Two independent methods were used to measure this parameter. The first method was essentially that described by Sasaki & Wagner (1971) with the exception that macro-aggregated iodinated (1311) human serum albumin was used as an alternative to microspheres. The majority of these particles had a diameter between 20 and 50 ,tm and consequently were trapped by intestinal capillaries so that the percentage of intracardiac administered macro-aggregated albumin collected in the jejunum could be taken as a measure of the percentage of cardiac output delivered to the jejunum. Nylon 00 tubing, filled with heparanized saline and attached to a blood pressure transducer, was used to cannulate the right carotid artery. The cannula was inserted into the left ventricle and its position verified by the large increase in the pulse pressure as the cannula passed the aortic valves. Saline was infused i.v. for 15 or 45 min after which 0-2 ml. (1 /,cc) macroaggregated albumin was injected into the left ventricle. When the effects of angiotensin were studied, saline was infused for 30 min followed by angiotensin for 15 min after which the macro-aggregated albumin was injected. The sac of jejunum was removed from the animal 10 min after the injection of macro-aggregated albumin, opened, damp blotted, weighed and assayed for 131I in a y-well scintillation counter. The second method was that of Sapirstein (1958) which depends on the observation that 86Rb is almost completely cleared from the plasma during its passage through an organ. In these experiments, 0-2 ml. (1 #uc) 86Rb Cl was injected into a femoral vein and 45 see later the intestine removed, blotted, weighed and assayed for 86Rb in a y-well scintillation counter.
Radioactivity assay Each 0-1 ml. sample of intestinal contents, containing [H3]inulin, was added to a counting vial containing 10 ml. scintillation fluid (0-8 % butyl PBD in 1: 1, v /v methanol :toluene) and counted in a Phillips automatic scintillation counter. Intestines, containing 86Rb or l3lI, were dissolved in conc. HNO4, made up to 5 ml. with distilled water and counted in a y-well scintillation counter. All counts were corrected for background and quenching to give d.p.m. Expression of results Fluid absorption is defined as the loss of fluid from the jejunum sac during the experiment and is recorded as ml./30 min per gram wet weight of sac. There is no significant change in the water content of the jejunum following the infusion of angiotensin, the wet/dry wt. ratio being 5-9 ± 0-4 (5) following the infusion of saline and 6-1 ± 0-3 (4) and 6-2 ± 0-7 after the infusion of 0-59 and 590 ng angiotensin/ kg per minute respectively. Transmural p.d. is taken as the mean p.d. recorded over a 30 min period. Student's t test was used to determine the significance of differences between means.
Materials Angiotensin (Hypertensin, Ciba) was diluted with sterile saline to a concentration of 10 jug/ml. and stored in sealed siliconized ampoules. Immediately before use, the angiotensin was diluted to a suitable concentration in isotonic saline. [3H]inulin 86RbCl and 13II-labelled macro-aggregated albumin were obtained from the Radiochemical Centre, Amersham.
ANGIOTENSIN ON INTESTINAL TRANSPORT
415
RESULTS
The use of non-absorbable markers to measure fluid transport has been well documented. In a comprehensive study, Miller & Schedl (1970) showed that less than 2 % of inulin was lost from an intestinal perfusate during a 3 hr test and concluded that this material is suitable for measuring fluid transport in preparations of small intestine. The effectiveness of inulin as a non-absorbable marker was confirmed in the modified preparation of jejunum used in these experiments. Fluid transport was
30O 4.1 41
4.1
0
0. co
CL C
-
L
4J
1-0io
120 60 90 Time (min) Fig. 1. The rate of fluid absorption, as a function of time, from sacs of rat jejunum in vivo. Each point represents the mean of two observations.
30
determined in a series of animals by both the inulin method and by the direct technique of measuring the loss of weight of the sacs. Over a period of 60 min, in eight preparations, fluid transport measured by the inulin method (1.48 + 0-10 ml./g wet wt. jejunum per hour) was not significantly different from that obtained by direct weighing (1.59 + 0'13 ml./g wet wt. jejunum per hour). Furthermore, in the presence of 30 mg/tOO ml. carrier inulin, 93-6 + 4-1 0/ of the inulin injected into the sac was recovered at the end of the experiment. The suitability of the preparation was further tested by measuring T.1
PHY 253
416
JENNIFER E. BOLTON AND OTHERS
V-
0 0
0 0
V
V
v
C)
C0
o0 CO
0
0-
.5
.--
0
0
COm
10
IV 0
0 C) 0
0l
10
10-
I-
.0 -Q
0
00 0
+1
+1
+1 0
0
W-
CO
0
pI fQ
+1 0 0
o
.--
444
.-10
t-
0 0
co 10
+l
+1
C6
C>
(M
'.
0
a44 0
CO
0 O
.;Z
W4 4
t-
~00
to
w
0
ODw
0 m
> C)
_
I
EM¢
m
o
0;0
3
+I
.--
~~~~~0 0
I-
02
w
-44
._
o -
Q
e
(D
PIZ
P.-
o MD
OD
(ID
GQ°
2
ANGIOTENSIN ON INTESTINAL TRANSPORT 417 the rate of fluid transport for varying periods to a maximum of 2 hr. The results of these experiments are given in Fig. 1 and show that the rate of fluid transport is constant for the first 60 min. After 60 min the rate of fluid absorption is reduced, presumably as a consequence of the small volume of fluid remaining in the sac and available for transport from this time. Having established the validity of using inulin as a non-absorbable marker to measure fluid transport from isolated sacs of rat jejunum and having shown that absorption is constant over a 60 min period, the following experiments were carried out to study the effects of angiotensin 10 4._
~.200
0
:g 150 0
L
_
Fig. 2. Dose dependent actions of angiotensin on fluid transport by rat jejunum in vivo. Angiotensin was infused during the second period and fluid transport is expressed as rate of transport in the second period as a percentage of that in the first (control) period. Bars represent s.E. of mean.
On fluid transport by this preparation. Fluid transport was measured over two consecutive 30 min periods. Isotonic saline was infused ix.V during the first 30 min period while saline or angiotensin in saline was
infused during the second period. In this way the first 30 min absorption period was used as a control for the second period. The results of these experiments are presented in Table 1. There was no significant difference between fluid absorption in the first and second 30 min periods in those experiments where saline was infused continuously, confirming the results presented in Fig. I that
